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. 2022 Jun 15;42(8):768–783. doi: 10.1002/cac2.12321

FIGURE 3.

FIGURE 3

Expression of p‐PSMA‐CAR‐NK92MI and PSMA+ cell line‐specific activation of p‐PSMA‐CAR‐NK92MI cells. (A) Schematic diagram of p‐PSMA‐CAR‐NK92MI with an extracellular antigen‐binding polypeptide region (p‐PSMA), a transmembrane region (CD244), an intracellular ITAM (CD244 and the NKG2D costimulatory domain) and a puromycin‐tag. (B) Western blotting analysis indicated positive expression of CAR structures by the lentivirus and p‐PSMA‐CAR‐NK92MI cells. (C) Assessment of expression of p‐PSMA and surface expression of CAR in NK92MI cells by flow cytometry. (D) CAR‐NK92MI cells could be specifically activated to perform tumor killing as exemplified by a cell lysis assay. NK92MI, p‐PSMA‐CAR‐NK92MI and anti‐meso‐CAR‐NK92MI cells were co‐cultured with C4‐2 cells (PSMA+/meso–), LNCaP cells (PSMA+/meso–), PC3 cells (PSMA–/meso–) and SKVO3 (PSMA–/meso+) cells at the indicated effector to target ratios for 4 hours. p‐PSMA‐CAR‐modified NK92MI cells could selectively and specifically kill PSMA+ target cells. (E) Quantitative data for the percentage of surface CD107a expression after different stimulations. The data are presented as the means ± SEMs from three independent experiments. *, P <0.05; **, P <0.01; ***, P <0.001; ****, P < 0.0001; ns, not significant. Abbreviations: CD244, lymphocyte activation molecule‐related receptor 2B4; ITAM, immunoreceptor tyrosine‐based activation motif; NKG2D, natural killer group 2 member D; CAR, chimeric antigen receptor; PSMA, prostate‐specific membrane antigen; meso, mesothelin; SEM, standard error of the mean.